多肽bCAT通过下调白念珠菌HWP1基因表达抑制生物被膜的形成机制

Effect of catestatin on inhibiting C. albicans biofilm formation by down-regulating the expression of HWP1

目的明确多肽bCAT抑制白念珠菌生物被膜形成能力,并探索其与黏附基因HWP1表达的关系。方法标准菌株白念珠菌ATCC10231和临床菌株作为研究对象,XTT法检测其生物被膜形成能力,bCAT抗浮游白念珠菌的MIC值以CLSI-M27-A3方法确定,XTT法及菌落计数检测bCAT抑制生物被膜形成作用,并计算代谢活性确定抑制50%生物被膜形成的MIC(BIC_(50)),bCAT减少白念珠菌的黏附作用经倒置显微镜下观察及菌落计数法检测,并采用RT-PCR通过2^(-ΔΔCt)法计算HWP1的表达量。统计方法为单因素方差分析,组内比较采用Dunnett T3检验。结果标准菌株及临床菌株均有较强生物被膜形成能力。bCAT抗浮游状态的白念珠菌MIC值为40~80μmol/L,BIC_(50)为80~160μmol/L;并且bCAT能减少白念珠菌的黏附作用,空白对照组菌落计数为(27 822.22±2 472.74)cfu,bCAT浓度为160、80、40、20、10μmol/L时的菌落个数分别为(5 355.55±1 264.03)cfu、(11 377.78±2 232.58)cfu、(17 488.89±1 136.27)cfu、(22 377.78±3 521.99)cfu、(26 044.44±1 329.57)cfu。组间差异具有统计学意义(F=147.018,P=0.001),组内比较发现160、80、40μmol/L处理组与不含bCAT时差异具有统计学意义(P<0.05)。RT-PCR发现160μmol/L处理组HWP1相对表达量为空白对照组的12.24%。结论 bCAT能有效抑制白念珠菌生物被膜形成,其机制可能与降低HWP1基因的表达从而减少白念珠菌的黏附有关。具有一定的临床前景。

Objective To investigate whether catestatin （bovine chromogranin A 344-364） can inhibit the biofilm formation of Candida albicans and examine its relationship with the expression of adhesion gene HWP1. Methods Clinical strains and standard strain ATCC 10231 of C. albicans were studied. XTT [2,3-bis-（2-methoxy-4-nitro-5-sulfophenyl）-2H-tetrazolium-5-carboxanilide] method was used to assess the ability of C. a[bicans biofilm formation. Antifungal activity against planktonic Candida ceils was evaluated in terms of minimum inhibitory concentrations （MICs） according to the description in CLSI-M27-A3. XTT assay and colony count were used to assess the effect of catestatin on inhibiting C. albicans biofilm formation. The lowest concentration showing 50 % inhibition on biofilm formation （BIC50） was decided by calculating the metabolic activity. The adhesion of C. albicans reduced by catestatin was visualized under an inverted microscope and quantified by colony count. The expression of HWP1 was analyzed by RT-PCR. One-way analysis of variance （ANOVA） and Dunnett＇s T3 test were used to compare the results. Results Clinical strains and standard strain ATCC 10231 of C. albicans showed strong ability in forming biofilm. Catestatin exhibited MICs ranging from 40 μmol/L to 80μmol/L against planktonic C. albicans cells, and BIC50 of 80-160 μmol/L in inhibiting C. albicans biofilm formation. Catestatin reduced the adhesion of C. albicans. The colony- forming unit （CFU） was 27 822.22±2 472.74 in blank control group, while the CFU was 5 355.55±1 264.03, 11 377.78±2 232.58, 17 488.89±1 136.27, 22 377.78±3 521.99, and 26 044.44±1 329.57 in the presence of 160, 80, 40, 20, and 10 μmol/L catestatin, respectively （F=147.018, P-0.001）. The difference between control group and 160, 80, and 40 μmol/L catestatin was statistically significant （P〈0.05）. RT-PCR found the expression of HWP1 in the presence of 160μmol/L catestatin was about 12.24 % of that in blank control group. Conclusions Catestatin can effectively prevent C. aIbicans biofilm formation. This effect may be related to the down-regulated expression of adhesion gene HWP1 by catestatin, which results in reduced adhesion of C. albicans. Promising clinical prospect is expected for this finding.